摘要
目的探究miR-5188在肝癌细胞中的作用及网络调控机制。方法过表达或敲低miR-5188表达后,检测肝癌细胞HepG2的增殖能力。利用miRNA靶基因预测数据库miRDB获取miR-5188潜在的靶基因,使用小干扰RNA(siRNA)敲低HepG2细胞中的上述靶基因后检测细胞增殖能力。采用实时荧光定量PCR法检测正常肝细胞LO2和HepG2细胞中miR-5188、pre-miR-5188和pri-miR-5188的表达水平,并分析长链非编码RNA NEAT1(下文简称NEAT1)、NONO/PSF复合体对pri-miR-5188的影响。结果过表达miR-5188后,HepG2细胞的增殖能力增强;敲低miR-5188表达后,HepG2细胞的增殖能力减弱,差异均有统计学意义(P均<0.05)。敲低DIDO1表达后,HepG2细胞的增殖能力增强;过表达DIDO1后,HepG2细胞的增殖能力减弱,差异均有统计学意义(P均<0.05)。过表达miR-5188后,HepG2细胞中DIDO1的表达水平降低;敲低miR-5188表达后,HepG2细胞中DIDO1的表达水平升高,差异均有统计学意义(P均<0.05)。双荧光素酶报告基因实验检测结果显示,miR-5188靶向DIDO1 mRNA的3'非翻译区(3'-UTR)。与LO2细胞相比,HepG2细胞中miR-5188和pre-miR-5188的表达水平均显著升高,而pri-miR-5188的表达水平显著降低,差异均有统计学意义(P均<0.05)。与LO2细胞相比,HepG2细胞中NEAT1的表达水平显著升高(P<0.05),而NONO和PSF的表达水平差异均无统计学意义(P均>0.05)。敲低NEAT1、NONO或PSF表达后,HepG2细胞中pri-miR-5188的表达水平均显著升高(P均<0.05)。敲除NEAT1后,HepG2细胞中pri-miR-5188的表达水平升高,NONO与PSF的相互作用减弱,并且NONO/PSF与pri-miR-5188的结合减少。结论HepG2细胞中NEAT1的表达水平升高,提高了NONO/PSF复合体对pri-miR-5188的加工水平,增强了miR-5188/DIDO1轴对HepG2细胞增殖能力的正向作用。
Objective This paper is to investigate the role of miR-5188 in liver cancer cells and its network regulation mechanism.Methods After overexpression or knockdown of miR-5188,the proliferation ability of HepG2 cell was detected.The potential target genes of miR-5188 were obtained using miRNA target gene prediction database miRDB,and the proliferation ability of HepG2 cells was detected after the above genes were knocked down using small interfering RNA(siRNA).Real-time fluorescence quantitative PCR was used to detect the expression of miR-5188,pre-miR-5188,and pri-miR-5188 in LO2 cells and HepG2 cells.The effects of long noncoding RNA NEAT1(hereinafter referred to as NEAT1)and NONO/PSF complex on pri-miR-5188 were analyzed.Results After overexpression of miR-5188,the proliferation ability of HepG2 cells is increased,and after knockdown of miR-5188,the proliferation ability of HepG2 cells is decreased,with statistically significant differences(P<0.05).After knockdown of DIDO1,the proliferation ability of HepG2 cells is increased,and after overexpression of DIDO1,the proliferation ability of HepG2 cells is decreased,with statistically significant differences(P<0.05).After overexpression of miR-5188,the expression of DIDO1 in HepG2 cells is decreased,and after knockdown of miR-5188,the expression of DIDO1 in HepG2 cells is increased,with statistically significant differences(P<0.05).The double luciferase reporter gene test results show that miR-5188 targets the 3'untranslated region(3'-UTR)of DIDO1 mRNA.Compared with LO2 cells,the expression of miR-5188 and pre-miR-5188 in HepG2 cells are increased,while the expression of pri-miR-5188 is decreased,with statistically significant differences(P<0.05).Compared with LO2 cells,the expression of NEAT1 in HepG2 cells is increased(P<0.05),while the expression of proteins NONO and PSF do not change(P>0.05).After knockdown of NEAT1,NONO or PSF,the expression of pri-miR-5188 in HepG2 cells is significantly increased(P<0.05).After knockout of NEAT1,the expression of pri-miR-5188 i
作者
全养雅
黎慧娟
陈仕
周艳
QUAN Yangya;LI Huijuan;CHEN Shi;ZHOU Yan(Department of Gastroenterology,Hunan Provincial Brain Hospital,Changsha 410007,China)
出处
《国际消化病杂志》
CAS
2023年第6期394-400,共7页
International Journal of Digestive Diseases
基金
湖南省卫计委科研计划项目(B20180176)。
